Pyruvate Facilitates FACT-Mediated γH2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma.

DNA repair confers the resistance of tumor cells to DNA-damaging anticancer therapies, while how reprogrammed metabolism in tumor cells contributes to such process remains poorly understood. Pyruvate kinase M2 isoform (PKM2) catalyzes the conversion of phosphoenolpyruvate to pyruvate and regulates the last rate-limiting step of glycolysis. Here it is shown that the glycolytic metabolite pyruvate enhances DNA damage repair by facilitating chromatin loading of γH2AX, thereby promoting the radiation resistance of glioma cells. Mechanistically, PKM2 is phosphorylated at serine (S) 222 upon DNA damage and interacts with FACT complex, a histone chaperone comprising SPT16 and SSRP1 subunit. The pyruvate produced by PKM2 directly binds to SSRP1, which increases the association of FACT complex with γH2AX and subsequently facilitates FACT-mediated chromatin loading of γH2AX, ultimately promoting DNA repair and tumor cell survival. Intriguingly, the supplementation of exogenous pyruvate can also sufficiently enhance FACT-mediated chromatin loading of γH2AX and promotes tumor cell survival upon DNA damage. The levels of PKM2 S222 phosphorylation correlate with the malignancy and prognosis of human glioblastoma. The finding demonstrates a novel mechanism by which PKM2-produced pyruvate promotes DNA repair by regulating γH2AX loading to chromatin and establishes a critical role of this mechanism in glioblastoma radiation resistance.

Mitochondrial PKM2 regulates oxidative stress-induced apoptosis by stabilizing Bcl2.

Pyruvate kinase M2 isoform (PKM2) catalyzes the last step of glycolysis and plays an important role in tumor cell proliferation. Recent studies have reported that PKM2 also regulates apoptosis. However, the mechanisms underlying such a role of PKM2 remain elusive. Here we show that PKM2 translocates to mitochondria under oxidative stress. In the mitochondria, PKM2 interacts with and phosphorylates Bcl2 at threonine (T) 69. This phosphorylation prevents the binding of Cul3-based E3 ligase to Bcl2 and subsequent degradation of Bcl2. A chaperone protein, HSP90α1, is required for this function of PKM2. HSP90α1's ATPase activity launches a conformational change of PKM2 and facilitates interaction between PKM2 and Bcl2. Replacement of wild-type Bcl2 with phosphorylation-deficient Bcl2 T69A mutant sensitizes glioma cells to oxidative stress-induced apoptosis and impairs brain tumor formation in an orthotopic xenograft model. Notably, a peptide that is composed of the amino acid residues from 389 to 405 of PKM2, through which PKM2 binds to Bcl2, disrupts PKM2-Bcl2 interaction, promotes Bcl2 degradation and impairs brain tumor growth. In addition, levels of Bcl2 T69 phosphorylation, conformation-altered PKM2 and Bcl2 protein correlate with one another in specimens of human glioblastoma patients. Moreover, levels of Bcl2 T69 phosphorylation and conformation-altered PKM2 correlate with both grades and prognosis of glioma malignancy. Our findings uncover a novel mechanism through which mitochondrial PKM2 phosphorylates Bcl2 and inhibits apoptosis directly, highlight the essential role of PKM2 in ROS adaptation of cancer cells, and implicate HSP90-PKM2-Bcl2 axis as a potential target for therapeutic intervention in glioblastoma.

PKM2 dephosphorylation by Cdc25A promotes the Warburg effect and tumorigenesis.

Many types of human tumour cells overexpress the dual-specificity phosphatase Cdc25A. Cdc25A dephosphorylates cyclin-dependent kinase and regulates the cell cycle, but other substrates of Cdc25A and their relevant cellular functions have yet to be identified. We demonstrate here that EGFR activation results in c-Src-mediated Cdc25A phosphorylation at Y59, which interacts with nuclear pyruvate kinase M2 (PKM2). Cdc25A dephosphorylates PKM2 at S37, and promotes PKM2-dependent ß-catenin transactivation and c-Myc-upregulated expression of the glycolytic genes GLUT1, PKM2 and LDHA, and of CDC25A; thus, Cdc25A upregulates itself in a positive feedback loop. Cdc25A-mediated PKM2 dephosphorylation promotes the Warburg effect, cell proliferation and brain tumorigenesis. In addition, we identify positive correlations among Cdc25A Y59 phosphorylation, Cdc25A and PKM2 in human glioblastoma specimens. Furthermore, levels of Cdc25A Y59 phosphorylation correlate with grades of glioma malignancy and prognosis. These findings reveal an instrumental function of Cdc25A in controlling cell metabolism, which is essential for EGFR-promoted tumorigenesis.